Cheng Yu-Che, Hsueh Chin-Chen, Lu Shao-Chun, Liao Ta-Hsiu
Department of Biochemistry and Molecular Biology, College of Medicine, National Taiwan University, Taipei, Taiwan.
Biochem J. 2006 Sep 1;398(2):177-85. doi: 10.1042/BJ20060564.
DNase II is an acid endonuclease that is involved in the degradation of exogenous DNA and is important for DNA fragmentation and degradation during cell death. In an effort to understand its catalytic mechanism, we constructed plasmids encoding nine different histidine (H)-to-leucine (L) mutants for porcine DNase II and examined the enzyme properties of the expressed mutant proteins. Of the mutants, all but H132L were secreted into the medium of expressing cells. Six of the mutated DNase II proteins (H41L, H109L, H206L, H207L, H274L and H322L) showed enzyme activity, whereas the H115L, H132L and H297L mutants exhibited very little activity. The H115L and H297L mutants were found to undergo correct protein folding, but were inactive. To further examine these mutants, we expressed H115A and H297A DNase II mutants; these mutants were inactive, but their DNase activities could be rescued with imidazole, indicating that His115 and His297 are likely to function as a general acid and a general base respectively in the catalytic centre of the enzyme. In contrast with the secreted mutants, the H132L mutant protein was found in cell lysates within 16 h after transfection. This protein was inactive, improperly folded and was drastically degraded via the proteosomal pathway after 24 h. The polypeptide of another substitution for His132 with lysine resulted in the misfolded form being retained in endoplasmic reticulum.
脱氧核糖核酸酶II（DNase II）是一种酸性内切核酸酶，参与外源DNA的降解，对细胞死亡过程中的DNA片段化和降解至关重要。为了了解其催化机制，我们构建了编码猪DNase II的9种不同组氨酸（H）到亮氨酸（L）突变体的质粒，并检测了表达的突变蛋白的酶学性质。在这些突变体中，除H132L外，其他所有突变体均分泌到表达细胞的培养基中。6种突变的DNase II蛋白（H41L、H109L、H206L、H207L、H274L和H322L）表现出酶活性，而H115L、H132L和H297L突变体的活性非常低。发现H115L和H297L突变体能够正确进行蛋白质折叠，但无活性。为了进一步研究这些突变体，我们表达了H115A和H297A DNase II突变体；这些突变体无活性，但它们的DNase活性可以用咪唑挽救，这表明His115和His297可能分别在酶的催化中心作为广义酸和广义碱发挥作用。与分泌型突变体不同，H132L突变蛋白在转染后16小时内出现在细胞裂解物中。该蛋白无活性、折叠不当，并在24小时后通过蛋白酶体途径被大量降解。用赖氨酸替代His132的另一种替代多肽导致错误折叠的形式保留在内质网中。
